AbstractHeritable variation in a gene’s expression arises from mutations impactingcis- andtrans-acting components of its regulatory network, with expression variation often derived fromtrans-regulatory mutations within species. Here, we investigate howtrans-regulatory mutations are distributed within the genome and within a gene regulatory network by identifying and characterizing 69 mutations withtrans-regulatory effects on expression of the same focal gene inSaccharomyces cerevisiae. Relative to 1766 mutations without effects on expression of this focal gene, we found that thesetrans-regulatory mutations were enriched in coding sequences of transcription factors previously predicted to regulate expression of the focal gene. However, over 90% of thetrans-regulatory mutations identified mapped to other types of genes involved in diverse biological processes including chromatin state, metabolism and signal transduction. Finally, we find that the genomic distribution of these new regulatory mutations significantly overlaps with the genomic distribution oftrans-regulatory variants segregating withinS. cerevisiae.